Conclusions
Our findings demonstrate that osteopontin is expressed in both M2- and M4-ipRGCs, challenging the notion of osteopontin as a marker exclusively for αRGCs. IHC double-labeling for osteopontin and melanopsin provides a novel method to identify and differentiate M2 ipRGCs from other subtypes. This will support the study of ipRGC physiology in a subtype -specific manner and may, for instance, foster research in the field of optic nerve injury.
Methods
Single-cell RNA (scRNA-seq) sequencing data from murine RGCs were analyzed to identify expression patterns of Spp1 across ipRGCs. Immunohistochemistry (IHC) was performed on retinal cryosections and flatmounts from C57BL/6J mice to characterize the localization of osteopontin across ipRGCs. Neurite tracing was employed to study dendritic morphology and identify individual ipRGC subtypes.
Purpose
Melanopsin-expressing intrinsically photosensitive (ip) retinal ganglion cells (RGCs) can be divided into six different subtypes (M1 - M6). Yet, specific markers exist for only some of these subtypes that could be employed to study the function of individual subtypes. Osteopontin (Spp1) marks αRGC, suggesting that, across ipRGCs, it would only mark the M4-ipRGC subtype (synonymous to ON-sustained αRGCs). Recent evidence suggests that osteopontin expression could spread to other ipRGC subtypes. Therefore, this study aims to characterize the expression pattern of osteopontin across ipRGC subtypes in mice.
Results
scRNA-seq analysis revealed Spp1 expression in two distinct clusters of ipRGCs. IHC confirmed osteopontin colocalization with neurofilament heavy chain, an established marker for αRGCs, including M4-ipRGCs. Spp1 immunoreactivity was moreover identified in one additional group of ipRGCs. By dendritic morphology and stratification, those cells were clearly identified as M2-ipRGCs. Conclusions: Our findings demonstrate that osteopontin is expressed in both M2- and M4-ipRGCs, challenging the notion of osteopontin as a marker exclusively for αRGCs. IHC double-labeling for osteopontin and melanopsin provides a novel method to identify and differentiate M2 ipRGCs from other subtypes. This will support the study of ipRGC physiology in a subtype -specific manner and may, for instance, foster research in the field of optic nerve injury.